DESCRIPTION: All living organisms must regulate the homeostasis of redox active copper ions that are both essential for life and potentially toxic. The overall hypothesis of this research is that the cellular accumulation and distribution of copper is precisely controlled by the actions of multiple copper "trafficking" factors that communicate and interact with one another. The research presented here utilizes the genetically well defined baker's yeast Saccharomyces cerevisiae to study intracellular pathways of copper trafficking. Two systems will be explored. One is represented by BSD2, encoding an endoplasmic reticulum protein that helps prevent the uncontrolled uptake of copper and other metals. The action of BSD2 on copper is mediated through the plasma membrane metal transporter, Smflp. The second copper trafficking system is represented by ATX1, encoding a small cytosolic copper binding protein. Our studies indicate that Atx1p delivers copper to Ccc2p, the yeast homologue to the human copper transporters affected in Wilson and Menkes diseases. The following specific aims will address how the accumulation and intracellular trafficking of copper ions in yeast is controlled by the BSD2-SMF1 pathway and by pathways involving ATX1. Aim 1: To understand the involvement of Smflp in the Bsd2p-medicated control of copper accumulation. The contribution of Smflp to copper accumulation will be studied through measurements of copper uptake. A series of molecular and biochemical studies will probe the effects of bsd2 mutations on the accumulation and stability of the Smf1p protein and its cellular localization. Aim 2: To understand the interactions between Atx1p and Ccc2p that govern copper trafficking. Biochemical and molecular genetic approaches will be used to test whether Atx1p and Ccc2p physically interact. Through mutagenesis studies, the Atx1p sequences necessary for activity in vivo and copper binding in vitro will be identified. The direct transfer of copper ions from Atx1p to Ccc2p will be investigated in a cell free system. Many of these studies will also employ the human homologues to ATX1 and CCC2, encoded by the HAH1 and Menkes and Wilson disease genes. Aim 3: To identify other downstream targets of ATX1: Two genetic screens in yeast will be exploited to identify factors other than Ccc2p that may serve as recipients for copper delivery by Atx1p. Overall, we believe that these studies in yeast will provide new important information regarding the complex pathways of copper trafficking in eukaryotes that serve to control the homeostasis of this important redox active metal.